The present invention relates to a method for ligating the carboxylic and the amino groups of one or two peptide segments through an amide bond where the functional groups of the segments are either minimally protected, partially protected, globally protected or not protected at all. More particularly, it relates to a method for ligating one peptide segment to itself or two peptide segments to each other by using a masked aldehyde ester incorporated onto the carboxylic group of a first peptide segment through an enzymatic coupling procedure, which masked aldehyde ester is then released in order that it may react with a .beta.-functionalized amino group of a second peptide segment to form a ring leading to an O to N-acyl rearrangement step which results in the formation of an amide bond between the peptide segments.
The synthesis of peptides or proteins has become highly efficient with the advances of the solid-phase peptide synthesis and recombinant DNA technology. Solid-phase peptide synthesis with the aid of automation and other mechanical devices can quickly produce a peptide of greater than 100 amino acids or a library of hundreds of short peptides. The recombinant DNA technology with an optimal expression system can produce proteins accurately and in large quantity. The ideal method of chemical ligation of peptide segments would incorporate both the efficiency of the solid-phase method to generate specific segments and the availability of proteins generated by the recombinant method. The combination of the two types of production of peptide segments would enable engineered proteins to contain unusual structures or nongenetic encoded amino acids by a specific ligation method.
A strong impediment to this approach is a lack of an efficient method for their synthesis. In particular, there is no effective chemical method to selectively couple two unprotected peptide segments to form an amide bond. In general, protecting groups are necessarily attached to nontarget functional groups on the first peptide segment prior to activation of the C-.alpha. of the carboxylic group by a coupling reagent and the consequent peptide bond formation with the N-.alpha. of the amino group of the second protected peptide segment. As a result, the developments of the various protecting group schemes have been the key for the conventional approach of ligating peptide segments.
However, the use of protected peptide segments is incompatible with the overall scheme of engineering proteins using proteins produced by means of recombinant DNA technology as a source. It also has limitations of being labor-intensive and unpredictable, partly due to the solubility and coupling difficulties of protected peptide segments. Often, large protected peptide segments are minimally soluble in even the most powerful polar aprotic solvents such as dimethylsulfoxide (DMSO) and dimethylforamide (DMF). The problem of insolubility in protected peptide segments has been addressed with limited success in several ways, including the use of (1) partial protecting group strategy which masks all side chains except those of Ser, Thr, and Tyr, and (2) minimal protecting group strategy which masks only thiol and amino side chains. Protecting groups used in all these approaches alter peptide conformations. This creates a difficult problem in the synthesis of large peptides, since folding and renaturation are required after the completion of the synthesis and removal of protecting groups. These limitations, coupled with the ease of obtaining proteins and protein domains through recombinant DNA technologies, have suggested the need to develop a new strategy for ligating unprotected peptides and proteins in order to engineer new proteins with unusual structures, architectures and functions.
Since protecting groups are the root of the problem, scientists have developed two ligation strategies in the past ten years which use unprotected segments. One of the methods requires the use of enzymes in the reverse proteolysis process in conjunction with a high content of water-miscible solvents. Although enzymatic synthesis has been successful with small peptides, enzymatic synthesis of large peptides has presented difficulties. The stringent criteria demanded by using high molar concentrations of peptide segments accompanied by rapid completion of the reverse proteolytic process without the attendant hydrolysis or transpeptidation have been prohibitive obstacles in the enzymatic synthesis of large peptides. Nevertheless, the use of enzymes in coupling unprotected peptide segments eliminates the necessity of activating the carboxylic group involved in the coupling reaction of the peptide segments. Furthermore, it also provides the ability to perform the reaction in an aqueous environment.
Another strategy uses a tricyclic aromatic template containing an aryl alcohol and a thiol to form an active ester with the carboxyl segment and a disulfide with the amino segment, respectively, in order to bring two unprotected peptide segments in close proximity with each other. Such positioning of the peptide segments enables them to undergo an O to N-acyl transfer reaction (Fotouhi, N. et al., 1989; Kemp, D. S. et at., 1991).
A problem with the currently accepted methods of protein synthesis which include both conventional liquid state and solid state peptide syntheses is that their application is limited to small straight chain peptide segments, whereas the need exists for such a method of synthesis to be available for long straight chain peptides, branched straight chain peptides and circular peptides.
It is an object of this invention to provide a method of ligation of two peptide segments from the group comprising, but not limited to, long straight chain peptides, branched straight lo chain peptides and circular peptides, without protecting the various functional groups and without activating the carboxyl group of a first peptide segment which will form a peptide bond with the amino group of a second peptide segment.
In addition, it is an object of this invention to provide a method of ligation of a peptide segment to a compound from the group comprising, but not limited to, DNA by means of incorporating a masked .alpha.-aldehyde ester on a carboxylic group and activating that group by releasing the aldehyde thus allowing the carboxylic group to interact with an amino group to form an amide bond. Ligating proteins or peptides to DNA can be useful in biological studies.
It is a further object of this invention that the method developed in this application will make circular proteins readily available for biochemical, biophysical, and therapeutic uses.
Another object of this invention is linking multiple copies of unprotected peptides or proteins to a scaffold or template by an amide to produce a branched protein. This application has broad utility. The present method provides a specific and stable conjugation for peptide/protein antigen to a carder, drug to a protein, reporter group to an antibody or enzyme, and many others.
Furthermore, it is an object of this invention to provide a high effective molarity for peptide bond formation through the efficient O to N-acyl transfer reaction.
It is an additional object of this invention to provide a versatile means of enzymatic coupling to activate a carboxylic group.
Finally, it is an object of this invention that the reactions required in the method of domain ligation may be run in one vessel in aqueous solution, require only pH changes, no intermediate purification steps and no harsh final deprotection, renaturation or disulfide bond formation.